The proposed study will focus on matrix vesicles (MVs) which will play a role in the mineralization of teeth and bones. MVs are sub-microscopic, extracellular, membrane-invested particles that initiate calcification of dentin, growth cartilage and developing bone. Our lab was involved in the first identification, isolation and characterization of MVs. We have provided evidence that MV phosphatases, including alkaline phosphatase (ALP), are involved in MV mineralization. Furthermore, these phosphatases are integral to the MV membrane where mineral first appears (at its inner surface) suggesting an important role for ALP and other components of the MV membrane in initiating calcification. Our specific aims are directed toward an increased understanding of the molecular and structural organization of the MV membrane and sap, with emphasis on identifying major constituitive proteins, further testing of the function of ALP in the calcification mechanism of isolated MVs, and studying the regulation of MV biogenesis in vitro by cultured chondrocytes and bone cells. Proposed new studies include: 1) studying the relationship of ALP to the MV membrane using ALP releasing agents coupled with EM cytochemical and immunocytochemical localization of ALP; 2) A "western" blot analysis to identify major constitutive proteins of MVs and to assign these proteins to either MV membrane or sap; 3) studying in vitro calcification of isolated rat MVs to examine the role of ALP in supporting calcification, as perturbed by inhibitors of ALP or ALP releasing agents or by ALP-enhancing agents such as triiodothyronine, 1,25-OH2-D3 and NaF; and 4) studies of MV bio- genesis in culture by MC3T3-El rat osteoblast cells. A comparison of MVs produced by MC3T3-E1 cells versus MVs from primary chondrocyte cultures as to yield of MVs, the presence of characteristic major proteins, and the ability to deposit 45Ca; and 5) testing the effect on MV biogenesis of possible regulatory factors such as stage of the cell cycle or ionophore- induced Ca2+ entry into MV producing cells. This is a fundamental study of the mechanism by which dental and skeletal forms of mineralization are initiated. New knowledge of matrix vesicle calcification can be applied to a broad range of topics including specific diseases in which abnormal calcification occurs.